Colorectal Carcinoma Cell Line HCT116 Research Articles

A potential requirement for Smad3 phosphorylation in Notch-mediated EMT in colon cancer.

Colorectal cancer (CRC) remains a challenging disease to treat due to several factors including stemness and epithelial to mesenchymal transition (EMT). Dysfunctional signaling pathways such as Notch and TGF-β contribute to these phenomena. We previously found that cells expressing constitutively active Notch1 also had increased expression of Smad3, an important member of the TGF-β signaling pathway. We hypothesized that Smad3, mediates the Notch-induced stemness and EMT observed in CRC cells. The human colorectal carcinoma cell line HCT-116, stably transduced with constitutively active Notch-1 (ICN) or a GFP-vector control was treated with different combinations of TGF-β1, DAPT (a Notch inhibitor), or SIS3 (a Smad3 inhibitor). Western blot analysis was performed to determine the effects of Smad3 stimulation and inhibition on Notch and potential downstream EMT-related targets, CD44, Slug and Snail. Smad3 inhibition induced a decrease in Notch1 and Notch3 receptor expression and effectively inhibited CD44, Slug, and Snail expression. Colosphere forming ability was also reduced in cells with inhibited Smad3. These results indicate a key role of TGF-β signaling in Notch1-induced tumorigenesis, and suggest a potential use for Smad3 inhibitors in combination with Notch1 inhibitors that are already in use for CRC treatments.

A potent bioactive fraction against colon cancer from Plectranthus vettiveroides.

This study was designed to investigate the anticancer efficacy of the organic leaf extracts of the plant, Plectranthus vettiveroides (P. vettiveroides), and to analyze the molecular mechanism of the anticancer activity. The leaf extracts were prepared by polarity-graded serial extraction of the dried leaf powder. The cytotoxic effect of the extracts was analyzed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The most active ethyl acetate extract was subjected to bioactivity-guided fractionation by column chromatography, which yielded a cytotoxic fraction designated as the P. vettiveroides fraction (PVF). The anticancer property of PVF was confirmed further by clonogenic assay. The mechanism of PVF-induced cell death was analyzed by flow cytometry and fluorescence microscopy. Additionally, the effects of PVF on apoptotic and cell survival pathways were analyzed using western immunoblot analysis. A bioactive fraction PVF, was isolated from the ethyl acetate leaf extract. PVF showed significant anticancer activity against colon cancer cells, whilst normal cells were comparatively less affected. PVF induced strong apoptotic stimuli in colorectal carcinoma cell line HCT116, involving both extrinsic and intrinsic pathways. Investigation into the molecular mechanism of anticancer activity of PVF in HCT116 cells revealed that the fraction activates the pro-apoptotic pathway via tumor suppressor protein 53 (p53) and inhibits the anti-apoptotic pathway by regulating phosphatidylinositol 3-kinase (PI3K) signaling. The findings of this study demonstrate, with mechanism-based evidence, the chemotherapeutic potential of a bioactive fraction PVF, derived from the leaves of the medicinal plant P. vettiveroides against colon cancer.

Design and characterization of PROTAC degraders specific to protein N-terminal methyltransferase 1

Protein N-terminal methylation catalyzed by N-terminal methyltransferase 1 (NTMT1) is an emerging methylation present in eukaryotes, playing important regulatory roles in various biological and cellular processes. Although dysregulation of NTMT1 has been linked to many diseases such as colorectal cancer, their molecular and cellular mechanisms remain elusive due to inaccessibility to an effective cellular probe. Here we report the design, synthesis, and characterization of the first-in-class NTMT1 degraders based on proteolysis-targeting chimera (PROTAC) strategy. Through a brief structure-activity relationship (SAR) study of linker length, a cell permeable degrader 1 involving a von Hippel-Lindau (VHL) E3 ligase ligand was developed and demonstrated to reduce NTMT1 protein levels effectively and selectively in time- and dose-dependent manners in colorectal carcinoma cell lines HCT116 and HT29. Degrader 1 displayed DC50 = 7.53 μM and Dmax > 90% in HCT116 (cellular IC50 > 100 μM for its parent inhibitor DC541). While degrader 1 had marginal cytotoxicity, it displayed anti-proliferative activity in 2D and 3D culture environment, resulting from cell cycle arrested at G0/G1 phase in HCT116. Label-free global proteomic analysis revealed that degrader 1 induced overexpression of calreticulin (CALR), an immunogenic cell death (ICD) signal protein that is known to elicit antitumor immune response and clinically linked to a high survival rate of patients with colorectal cancer upon its upregulation. Collectively, degrader 1 offers the first selective cellular probe for NTMT1 exploration and a new drug discovery modality for NTMT1-related oncology and diseases.

Hyal1 Expression in Colorectal Carcinoma Cell Migration and Invasiveness: Significance and Mechanism.

Objective To clarify the significance of hyaluronidsase 1 (Hyal1) expression in colorectal carcinoma (CRC) and its impact on tumor cell migration and invasiveness. Methods Human CRC cell lines SW480, HCT116, and SW620 were purchased, ELISA and western blot were used to detect the expression of Hyal1 in cells, CCK-8 assay to detect cell proliferation ability, cell scratch assay to check cell migration rate, and cell invasion was detected by the transwell assay. The correlation of Hyal1 with CRC cell migration and invasiveness capacities was analyzed. Result ELISA results showed that supernatant Hyal1 level was the lowest in SW480, highest in HCT116, with the level in SW620 in between (P < 0.05). No evident difference was identified by western blot in Hyal1 protein expression among the three cells (P > 0.05). The cell scratch assay and transwell assay showed that the migration and invasion ability of HCT116 cells was higher than that of SW620 (P < 0.05). In vitro, Hyal1 had a synergistic relationship with the invasiveness and migration capacities of CRC cells (P < 0.05). Conclusion Hyal1 is elevated in CRC and is consistent with the invasiveness and metastasis abilities of CRC cells. It is hoped that this research can provide reference for future prevention and treatment of CRC.

Camptothecin, triptolide, and apoptosis inducer kit have differential effects on mitochondria in colorectal carcinoma cells.

Mitochondrial fission and fusion are required for cell survival, and several studies have shown an imbalance between fission and fusion in cancer. High levels of mitochondrial fusion are observed in drug‐resistant tumor cells, whereas mitochondrial fission may be important in sensitizing tumor cells to chemotherapy drugs. Based on current knowledge, we hypothesized that different chemotherapeutics might differentially affect mitochondrial dynamics and energy production. Thus, we selected chemotherapeutics with different mechanisms of action (camptothecin, triptolide and apoptosis inducer kit) and investigated their effect on mitochondria in colorectal carcinoma cells. We report that these chemotherapeutics decreased the activity of complex I and reduced the mitochondrial membrane potential, and also decreased the size of mitochondria in the colorectal carcinoma cell lines DLD1 and HCT‐116. Treatment with camptothecin, triptolide and/or apoptosis inducer kit results in differential effects of fission on apoptosis in these cells. Our results suggest that fission is an important process in apoptosis induced by chemotherapeutics.

Modeling and measuring glucose diffusion and consumption by colorectal cancer spheroids in hanging drops using integrated biosensors

As 3D in vitro tissue models become more pervasive, their built-in nutrient, metabolite, compound, and waste gradients increase biological relevance at the cost of analysis simplicity. Investigating these gradients and the resulting metabolic heterogeneity requires invasive and time-consuming methods. An alternative is using electrochemical biosensors and measuring concentrations around the tissue model to obtain size-dependent metabolism data. With our hanging-drop-integrated enzymatic glucose biosensors, we conducted current measurements within hanging-drop compartments hosting spheroids formed from the human colorectal carcinoma cell line HCT116. We developed a physics-based mathematical model of analyte consumption and transport, considering (1) diffusion and enzymatic conversion of glucose to form hydrogen peroxide (H2O2) by the glucose-oxidase-based hydrogel functionalization of our biosensors at the microscale; (2) H2O2 oxidation at the electrode surface, leading to amperometric H2O2 readout; (3) glucose diffusion and glucose consumption by cancer cells in a spherical tissue model at the microscale; (4) glucose and H2O2 transport in our hanging-drop compartments at the macroscale; and (5) solvent evaporation, leading to glucose and H2O2 upconcentration. Our model relates the measured currents to the glucose concentrations generating the currents. The low limit of detection of our biosensors (0.4 ± 0.1 μM), combined with our current-fitting method, enabled us to reveal glucose dynamics within our system. By measuring glucose dynamics in hanging-drop compartments populated by cancer spheroids of various sizes, we could infer glucose distributions within the spheroid, which will help translate in vitro 3D tissue model results to in vivo.

Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma.

Background: p53 mutations are highly frequent in various human cancers and are reported to contribute to tumor malignance and chemoresistance. In this study, we explored the mechanism by which mutant p53 promotes carcinogenesis and chemoresistance and provided novel insights into cancer therapy. Materials and methods: A total of 409 patients with colorectal carcinoma from TCGA database were subdivided into two groups according to the p53 status, namely, mutant p53 and wild-type p53, following with GSEA analysis. The differences of the clinicopathologic index were also analyzed. Two HCT116 cell lines containing hot spots at codons R273H and R248W of p53 were constructed based on HCT116 with knockout p53, respectively. Cell viability, mobility, clonogenesis, and stemness were detected by CCK8, transwell migration and invasion, colonogenic, and sphere formation assays. Resistance to 5-FU was examined by live-dead staining and flow cytometry. qPCR, Western blot, and luciferase reporter assay were performed to identify that deficient or mutant p53 promoted chemoresistance of the colorectal carcinoma cell line HCT116 through the TCF21/CD44 signaling pathway, with the following rescue assays by overexpression of TCF21 and knockdown of CD44. Results: Patients with recurrence harbor a higher frequency of mutant p53 than those without recurrence (p < 0.05). The mutant p53 group developed a larger tumor than the wild-type one. GSEA analysis showed that oncogenic signatures were enriched in the mutant p53 group. Extracellular assays showed that cancer cells with deficient or mutant p53 (R273H and R248W, respectively) promoted colon cancer cell growth, migration, invasion, and stemness. The mutant cancer cells were also observed to be significantly resistant to 5-FU. Xenografts also confirmed that HCT116 cells harboring deficient or mutant p53 promoted cancer growth and 5-FU tolerance. Luciferase reporter assay showed that deficient or mutant p53 R237H and R248W endowed cancer cells with chemoresistance by activating CD44 via repressing the nuclear transcription factor TCF21 expression. Overexpression of TCF21 or knockdown of CD44 could rescue the sensitivity to 5-FU in deficient and mutant p53 HCT116 cell lines. Conclusion: Our results, for the first time, reveal a novel deficient or mutant p53/TCF21/CD44 signaling pathway which promotes chemoresistance in colorectal carcinoma. The axis could be an effective therapeutic strategy against deficient- or mutant p53-driven chemoresistance.

The Chemosensitizing Effect of Portulaca oleracea Methanol Extract on Human Colorectal and Lung Cancer Cells Against Irinotecan

Cancer is a major health problem and it has been causing great damage in recent years worldwide. Serious side effects of chemotherapeutics used in cancer treatment make natural products valuable. Combined use of chemotherapy drugs and herbal products is an important approach in reducing side effects and revealing a more effective treatment. In this study, the effects of methanolic extract from Portulaca oleracea (POM) alone and in combination with Irinotecan were investigated against human colorectal carcinoma cell line HCT-116 and human lung adenocarcinoma cell line A549. The antiproliferative/anti-colony formation effects were determined using WST-1 assay and clonogenic assay. Cell death detection Elisa assay was employed to exhibite the triggered apoptosis, and quantitative real time polimerase chain reaction (qRT-PCR) was performed to evaluate the mRNA expression of mTOR, Akt and PTEN genes, which are significant modulators of apoptotic and survival pathways. It was determined that 72 h treatment with increasing concentrations of POM (0-1000 mg mL-1) decreased the viability of HCT116 and A549 cells in a dose-dependent manner. Also, POM and Irinotecan exhibited synergistic effects in both cell lines. The combination of POM and Irinotecan suppressed the colony forming capacity of HCT116 and A549 cells by 84% and 79%, respectively. POM also altered mTOR, Akt and PTEN gene expressions in HCT-116 and A549 carcinoma cells, both alone and in combination with Irinotecan. This study shows that the phytotherapeutic effect of P. oleracea needs to be studied more intensively.

Trafficking of Full-Length and N-Terminally Truncated Cathepsin B in Human Colorectal Carcinoma Cells

Cathepsin B is an endo-lysosomal cysteine protease. However, its increased expression and altered localization to the extracellular space, to mitochondria, or to the nucleus has been linked to tumor progression. In the present study, we show enhanced levels of cathepsin B in adenocarcinoma tissue in comparison to adjacent normal colon. Additionally, cathepsin B was observed in the nuclear compartment of mucosal cells in adenocarcinoma tissue samples and in the nuclei of the colorectal carcinoma cell line HCT116. Accordingly, a distinct 40-kDa form of cathepsin B was detected in HCT116 cells, which is proposed to represent a specific form lacking the signal peptide and parts of the propeptide. Trafficking studies with an EGFP-tagged N-terminally truncated form, mimicking the 40-kDa form, demonstrated accumulation in aggresome-like inclusion bodies, while EGFP-tagged full-length cathepsin B revealed regular sorting to endo-lysosomes. We conclude that the identity of nuclear cathepsin B in colorectal adenocarcinoma (in situ) and in carcinoma cells (in vitro) cannot be attributed to either full-length or 40-kDa N-terminally truncated cathepsin B forms. Hence, future studies are needed to demonstrate which form/s of cathepsin B may be sorted to the nuclei of colorectal carcinoma cells, and whether redundant regulation of related cathepsin expression occurs.

Design, Synthesis, Characterization, and Crystal Structure Studies of Nrf2 Modulators for Inhibiting Cancer Cell Growth In Vitro and In Vivo.

Nrf2 is one of the important therapeutic targets studied extensively in several cancers including the carcinomas of the colon and rectum. However, to date, not many Nrf2 inhibitors showed promising results for retarding the growth of colorectal cancers (CRCs). Therefore, in this study, first, we have demonstrated the therapeutic effect of siRNA-mediated downmodulation of Nrf2 on the proliferation rate of CRC cell lines. Next, we have designed, synthesized, characterized, and determined the crystal structures for a series of tetrahydrocarbazoles (THCs) and assessed their potential to modulate the activity of Nrf2 target gene NAD(P)H:quinone oxidoreductase (NQO1) activity by treating colorectal carcinoma cell line HCT-116. Later, the cytotoxic potential of compounds was assessed against cell lines expressing varying amounts of Nrf2, viz., breast cancer cell lines MDA-MB-231 and T47D (low functionally active Nrf2), HCT-116 (moderately active Nrf2), and lung cancer cell line A549 (highly active Nrf2), and the lead compound 5b was tested for its effect on cell cycle progression in vitro and for retarding the growth of Ehrlich ascites carcinomas (EACs) in mice. Data from our study demonstrated that among various compounds 5b exhibited better therapeutic index and retarded the growth of EAC cells in mice. Therefore, compound 5b is recommended for further development to target cancers.

Antitumour Activity of Muricatacin Isomers and its Derivatives in Human Colorectal Carcinoma Cell HCT116.

Colorectal cancer is one of the leading causes of cancer-related death in elderly people. The natural product muricatacin is an important member of the γ-lactone family, and it has exhibited antitumour activity in multiple cancer cell lines; however, the antitumour activities of muricatacin stereoisomers and their derivatives in colorectal cancer cells have not yet been systematically explored. The colorectal carcinoma cell line HCT116 was investigated in this study. Cell proliferation was assessed by MTT assay or crystal violet staining. Cell cycle arrest and cell apoptosis were evaluated by flow cytometry assay. The expression levels of p53, p21, cyclin E, cyclin D1, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9 and LC3B were measured using western blot analysis. Autophagy induced by M2 was monitored by immunofluorescence assay with an antibody against LC3B. Cell proliferation assays showed that both naturally occurring muricatacin (M4) and its synthetic stereoisomer (M2) are potent cell growth inhibitors in HCT116 cells, with IC50 values of 79.43 and 83.17μM, respectively; these values are much lower than those of the other two isomers, M1 and M3, and those of the sixmembered lactone analogues. The flow cytometry analysis revealed that M2 and M4 induced significant cell cycle arrest during G0/G1 phase and caused relatively low apoptosis rates in HCT116 cells. Further analysis indicated that M2 caused p53-independent p21 induction and cyclin E/cyclin D1 downregulation. In addition, M2 also markedly induced autophagy in the early stage of administration. Our results suggested that muricatacins possess potent antitumour activity against the colorectal carcinoma cell line HCT116 through inducing G0/G1 phase cell cycle arrest and autophagy in the early stage of administration.

Abstract 5249: The transcriptional regulation of CCND1 by miR-15a-5p in the nucleus of colorectal carcinoma cell lines

Abstract Although miRNAs are important post-transcriptional regulators of gene expression in cytoplasm, more and more evidences demonstrate that miRNAs also have nuclear functions. The majority of miRNAs are present in both the cytoplasm and the nucleus. The canonical function of translational repression in cytoplasm is well studied, but the functions of nuclear miRNAs are currently not well understood. From previous studies, we found that miR-15a-5p (miR-15a for short) can be detected in the nucleus of the colon cancer cell line. By transfected miR-15a labelled with biotin, we validated that miR-15a could bind to DNA (especially dsDNA) in the nucleus of colorectal carcinoma cell line HCT116. To find the targets of miR-15a in the nucleus, we scanned the promoter region of the potential targets which were predicted by Targetscan, miRWalk, and miRDB. The promoter of CCND1 has one region that is complementary to miR-15a. By integrating the RNA-seq data and miRNA-Seq data from NCI60 cell line panel and TCGA (The Cancer Genome Atlas), we found that CCND1 was negatively regulated by miR-15a in colorectal cancer. To confirm that miR-15a regulates CCND1’s transcription in the nucleus, we constructed the promoter of CCND1 and a luciferase reporter into one plasmid, then site-specific mutation was introduced into the potential binding site of miR-15a in the promoter of CCND1. The luciferase assay demonstrated that the mutation in the binding site decreased the expression of CCND1, which validate that miR-15a may regulate the expression of CCND1 by binding to the promoter of CCND1 in the nucleus. The next step, we will further study the function of nuclear miR-15a distinct from the canonical paradigm in carcinogenesis. Note: This abstract was not presented at the meeting. Citation Format: Zhenzhen Liu, Xue Bai, Chunyan Li. The transcriptional regulation of CCND1 by miR-15a-5p in the nucleus of colorectal carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5249.

Synthesis and Biological Activity of Peptide α-Ketoamide Derivatives as Proteasome Inhibitors.

Proteasome activity affects cell cycle progression as well as the immune response, and it is largely recognized as an attractive pharmacological target for potential therapies against several diseases. Herein we present the synthesis of a series of pseudodi/tripeptides bearing at the C-terminal position different α-ketoamide moieties as pharmacophoric units for the interaction with the catalytic threonine residue that sustains the proteolytic action of the proteasome. Among these, we identified the 1-naphthyl derivative 13c as a potent and selective inhibitor of the β5 subunit of the 20S proteasome, exhibiting nanomolar potency in vitro (β5 IC50 = 7 nM, β1 IC50 = 60 μM, β2 IC50 > 100 μM). Furthermore, it significantly inhibited proliferation and induced apoptosis of the human colorectal carcinoma cell line HCT116.

Chemical Analysis and Cytotoxic and Cytostatic Effects of Twelve Honey Samples Collected from Different Regions in Morocco and Palestine.

The aim of this in vitro study is to characterize the phenolic compounds of twelve honey samples collected from different locations in Palestine (H1-6) and Morocco (H7-12) and to evaluate their cytotoxic and cytostatic effects in cells from the human colorectal carcinoma cell line HCT-116 and breast cancer cell line MCF-7. Quantitative HPLC analysis revealed nine phenolic compounds in three Moroccan honey samples, namely, syringic acid, tannic acid, caffeic acid, ferulic acid, coumaric acid, gallic acid, rosmarinic acid, epicatechin, and pyrogallol. Syringic acid, abundant in numerous types of honey with strong antioxidant capacities, was present at values ranging between 0.10 mg/100 g and 1.24 mg/100 g of Daghmos (H11) and Kabbar (H10) samples, respectively. No significant reductions in cell viability were observed in both cell lines treated with the Palestinian samples as measured with MTT assay. Significant cytostatic effects were after treatment of HCT cells with Morar honey H1 with IC50 of 1789 μg/ml. Three Moroccan samples, H7 (Zaâtar), H9 (Bochnikha), and H10 (Kabbar), showed slight, but significant cytostatic effects in HCT cells. A strong correlation was observed between cytostatic activity of MCF cells and antioxidant content (phenols, flavonoids, and flavonol). Furthermore, a strong negative correlation was detected between the cytostatic activity in HCT cells and the contents of syringic acid (r= -0.756) and tannic acid (r= -0.610). These results indicate that the traditionally known anticancer effects of honey might be mediated in part through cytostatic effects.

Targeting scFv-Fc-scTRAIL fusion proteins to tumor cells.

Fusion proteins combining hexavalent TRAIL with antibody fragments allow for a targeted delivery and efficient apoptosis induction in tumor cells. Here, we analyzed scFv-Fc-scTRAIL molecules directed against EGFR, HER2, HER3, and EpCAM as well as an untargeted Fc-scTRAIL fusion protein for their potentials to induce cell death both in vitro and in a xenograft tumor model in vivo. The scFv-Fc-scTRAIL fusion protein directed against EGFR as well as the fusion protein directed against EpCAM showed targeting effects on the two tested colorectal carcinoma cell lines Colo205 and HCT116, while a fusion protein targeting HER3 was more effective than untargeted Fc-scTRAIL only on Colo205 cells. Interestingly, another anti-HER3 scFv-Fc-scTRAIL fusion protein exhibiting approximately 10-fold weaker antigen binding as well as the HER2-directed molecule were unable to increase cytotoxicity compared to Fc-scTRAIL. A comparison of EC50 values of cell death induction and antigen binding supports the assumption that high affinity antigen binding is one of the requirements for in vitro targeting effects. Furthermore, a minimal number of expressed target antigens might be required for increased cytotoxicity of targeted compared to non-targeted molecules. In a Colo205 s.c. xenograft tumor model, strongest antitumor activity was observed for the anti-HER3 scFv-Fc-scTRAIL fusion protein based on antibody 3-43, with complete tumor remissions after six twice-weekly injections. Surprisingly, a similar in vivo activity was also observed for untargeted Fc-scTRAIL in this tumor model, indicating that additional factors contribute to the potent efficacy of targeted as well as untargeted hexavalent Fc-scTRAIL fusion proteins in vivo.

Mass Spectrometric Characterization of Peptides Associated with Molecules of the Major Histocompatibility Complex

Mass Spectrometric Characterization of Peptides Associated with Molecules of the Major Histocompatibility Complex

High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma.

Previous studies have found that G-protein-coupled receptor 116 (GPR116) is a regulator of breast cancer metastasis. However, the role of GPR116 in colorectal carcinoma (CRC) carcinogenesis and progression is unknown. In this study, We found GPR116 expression was significantly up-regulated in CRC specimens compared with corresponding non-cancerous tissues. Increased GPR116 expression in CRC was correlated with histological differentiation and distant metastasis. In addition, high expression of GPR116 was significantly associated with poor overall survival of CRC patients, which was also confirmed by GSE14333, GSE17536 and GSE33113 datasets from the Gene Expression Omnibus (GEO). Furthermore, we demonstrated that the ability of proliferation and invasion of CRC cell lines HCT116 and LOVO was markedly reduced after transfected with siRNA-GPR116. Meanwhile, GPR116 may drive EMT in CRC cells through AKT/EKR signaling pathway, resulting in metastasis. Thus, GPR116 may be a novel reliable prognostic indicator and a risk factor in CRC progression.

Overexpression of NOX4 predicts poor prognosis and promotes tumor progression in human colorectal cancer.

NADPH oxidase 4 (NOX4), a major source of reactive oxygen species (ROS) production, has been increasingly reported to be involved in tumorigenesis and/or tumor progression, but limited data are available regarding the role of NOX4 in colorectal carcinoma (CRC). We retrieved six independent investigations from Oncomine database and found that NOX4 is highly expressed in CRC tissues compared with corresponding normal controls. Similar results were also found in clinical specimens at both mRNA and protein levels. Immunohistochemical analysis indicated that NOX4 overexpression was highly correlated with T classification, N classification, distant metastasis, and poor prognosis of CRC patients, which was also confirmed by GSE14333 and GSE17536 datasets from the Gene Expression Omnibus. Furthermore, we demonstrated that when NOX4 expression was knocked down by siRNAs, cell proliferation, cell-cycle and apoptosis, migration and invasion were significantly altered in CRC cell lines HCT116 and LOVO. Meanwhile, NOX4 promoted cancer cell proliferation and apoptosis, migration and invasion by regulating the expression of relevant genes. By these approaches we aim to elucidate NOX4 may be a reliable prognostic factor or therapeutic target in CRC.

Downregulation of RASAL2 promotes the proliferation, epithelial-mesenchymal transition and metastasis of colorectal cancer cells.

RAS protein activator like 2 (RASAL2) is a RAS-GTPase-activating protein and has recently been identified to be a tumor suppressor in various types of human cancer; however, the function of RASAL2 in colorectal carcinoma (CRC) remains unclear. In the present study, the function of RASAL2 in CRC cells was investigated using a RASAL2 loss-of-function cell model. RASAL2 short hairpin RNA was transfected into the human CRC cell lines LoVo, SW620 and HCT116, and the wild-type colon cell line NCM460. The subsequent downregulation of RASAL2 was evaluated using western blot and reverse transcription-quantitative polymerase chain reaction analyses. It was observed that RASAL2 expression was significantly decreased in human CRC tissues and cell lines (P<0.01). In the loss-of-function cell models, RASAL2 expression was decreased significantly, while cell proliferation, colony formation, migration and invasion were increased (all P<0.01). These effects were associated with the induction of epithelial-mesenchymal transition and Raf/mitogen-activated protein kinase hyperactivation. The results of the present study indicate that RASAL2 is a potential therapeutic target to inhibit CRC progression and metastasis.

Multifunctional Hyaluronic Acid and Chondroitin Sulfate Nanoparticles: Impact of Glycosaminoglycan Presentation on Receptor Mediated Cellular Uptake and Immune Activation.

Hyaluronic acid (HA) and chondroitin sulfate (CS) polymers are extensively used for various biomedical applications, such as for tissue engineering, drug delivery, and gene delivery. Although both these biopolymers are known to target cell surface CD44 receptors, their relative cellular targeting properties and immune activation potential have never been evaluated. In this article, we present the synthesis and characterization of novel self-assembled supramolecular HA and CS nanoparticles (NPs). These NPs were developed using fluorescein as a hydrophobic component that induced amphiphilicity in biopolymers and also efficiently stabilized anticancer drug doxorubicin (DOX) promoting a near zero-order drug release. The cellular uptake and cytotoxicity studies of these NPs in different human cancer lines, namely, human colorectal carcinoma cell line HCT116 and human breast cancer cell line MCF-7 demonstrated dose dependent cytotoxicity. Interestingly, both NPs showed CD44 dependent cellular uptake with the CS-DOX NP displaying higher dose-dependent cytotoxicity than the HA-DOX NP in different mammalian cells tested. Immunological evaluation of these nanocarriers in an ex vivo human whole blood model revealed that unlike unmodified polymers, the HA NP and CS NP surprisingly showed platelet aggregation and thrombin-antithrombin complex formation at high concentrations (0.8 mg/mL). We also observed a clear difference in early- and late-stage complement activation (C3a and sC5b-9) with CS and CS NP triggering significant complement activation at high concentrations (0.08-0.8 mg/mL), unlike HA and HA NP. These results offer new insight into designing glycosaminoglycan-based NPs and understanding their hematological responses and targeting ability.